Maintenance of extrecellular fluid volume homeostasis is essential for hemodynamic stability, and abnormalities of renal sodium handling have been linked to cardiovascular disease and hypertension. Ultimate regulation of sodium excretion occurs in the distal nephron via conductive transport through the amiloride sensitive epithelial Na+ channel (ENaC). ENaC expression and activity in the apical membrane of epithelial cells is the rate limiting step in Na+ reabsorption not only in the kidney collecting duct, but in airway epithelia and colon as well. Aldosterone is the major regulator of ENaC expression and activity in responsive epithelia. The long term objectives of this research are to define the mechanisms through which mineralocorticoid hormones regulate vectorial sodium transport in responsive epithelia and studies are designed to examine the mechanisms of aldosterone regulation of ENaC. Work in the current grant period has identified a specific methyltransferase which activates ENaC and knockdown of this protein blocks the early aldosterone response. Studies are now proposed to determine if this protein is required for early aldosterone action via genomic or non-genomic pathways and determine how aldosterone regulates its activity. We have demonstrated that ENaC trafficks to the apical membrane of epithelial cells in lipid rafts and developed a model of ENaC trafficking in cultured epithelial cells. Cells deprived of aldosterone develop a loss of Na+ reabsorptive activity along with a loss of the cellular recycling compartment for ENaC. The recycling compartment and Na+ reabsorptive phenotype are restored over time by aldosterone. Restoration of the ENaC recycling pathway is associated with adlosterone up-regulation of multiple proteins in the endosomal compartment. We will examine the mechanisms by which aldosterone regulates the apical expression of ENaC from endosomal and/or recycling pathways and determine what specific aldosterone-regulated proteins control the activity and expression of ENaC in endosomes and exocyst compartments to support apical translocation of ENaC in Na+ reabsorptive states. PUBLIC HEALTH RELEVANCE: Aldosterone is the major hormone which regulates sodium reabsorption in the kidney in mammals. Abnormalities of sodium handling can lead to diseases such as hypertension and heart failure and have been implicated in the progression of cystic fibrosis lung disease. These studies are designed to determine the cellular mechanisms by which aldosterone regulates sodium reabsorption in kidney cells.